The majority of cancer deaths are caused by blood-borne metastasis from a primary epithelial tumor, but our understanding of this process is limited. Epithelial-to-Mesenchymal Transition (EMT) is a fundamental developmentally regulated change in cell fate, whose aberrant activation in cancer has been proposed as contributing to the invasiveness and motility of cancer cells. However, given the difficulty in studying human cancer metastasis, most studies have relied on cell line and mouse models, and the relevance of EMT in human cancer is not well established. New technologies enabling the isolation and molecular analysis of circulating tumor cells (CTCs), rare cancer cells that are in transit through the bloodstream, now provide a unique opportunity to define mechanisms involved in human cancer metastasis. We propose a molecular analysis of CTCs to validate the prevalence of EMT in human breast cancer, use a carefully titrated in vitro model of EMT to identify key effectors that could constitute potential drug targets, and test their effectiveness using a mouse model in which CTC quantitation provides a rapid readout for the metastatic potential of human breast cancer cells. Our approach has three aims: in Aim 1, we will determine whether EMT is a consistent feature of different histological subtypes of breast cancer and whether these markers evolve dynamically during response or resistance to therapy. This will be accomplished using RNA-in-situ probes that we have developed, capable of scoring quantitative epithelial and mesenchymal markers within individual cells. RNA sequencing will then be applied to identify EMT associated transcripts within breast circulating tumor cells, thus providing insight into the relevant biological pathways. In Aim 2, we will study an inducible in vitro model of EMT, which effectively mediates a regulated epigenetic switch. We have generated timelines of both transcriptionaland chromatin immuno-precipitation profiles, which will be used to identify candidate effectors of EMT, seeking potential drugable targets. In Aim 3, we will establish a robust mouse assay to monitor the ability of breast cancer cells to metastasize and thus test potential suppressors of EMT-related phenotypes. CTC enumeration in the mouse model will be used as a rapid and quantifiable readout for vascular invasiveness. Together, these experiments aim to combine novel molecular and bioengineering technologies to address the relevance of EMT as a therapeutic target in suppressing human cancer metastasis.